Tài liệu Báo cáo khoa học: FOXM1c transactivates the human c-mycpromoter directly via the two TATA

FOXM1c transactivates the human c-myc promoter

directly via the two TATA boxes P1 and P2

Inken Wierstra1 and Ju¨rgen Alves2

1 Institute of Molecular Biology, Medical School Hannover, Germany

2 Institute of Biophysical Chemistry, Medical School Hannover, Germany

c-Myc, a key regulator of proliferation, differentiation

and apoptosis, plays a central role in cell growth

control and can induce quiescent cells to enter into

S-phase [1–7]. Because c-Myc potently stimulates pro￾liferation and inhibits differentiation it possesses a high

transformation potential that is supplemented by its

cell growth and angiogenesis-promoting, cell-adhesion￾reducing, immortality and genomic-instability-causing

activities. c-myc expression correlates strictly with cell

proliferation. c-Myc regulates target genes either by

activation via E-boxes or by repression via initiator

(Inr)-dependent and Inr-independent mechanisms.

c-Myc acts as part of the Myc ⁄Max ⁄Mad network in

which Max is the heterodimerization partner for

c-Myc and Mad proteins, the c-Myc antagonists,

which repress target genes via E-boxes.

The forkhead ⁄ winged helix transcription factor

FOXM1, expression of which correlates strictly with

proliferation, stimulates proliferation by promoting

S- and M-phase entry and regulates genes that control

G1 ⁄ S and G2 ⁄M transition [8–27]. The activity of

FOXM1 as a conventional transcription factor is

increased by proliferation signals and reduced by anti￾proliferative signals. Furthermore, FOXM1 is assumed

to be implicated in tumorigenesis [18,23–26,28].

We have previously shown that as a conventional

Keywords

c-myc; core promoter; FOXM1; TATA box;

TATA-binding protein

Correspondence

I. Wierstra, Wißmannstr. 17, D-30173

Hannover, Germany

Fax: +49 511 883 536

Tel. +49 511 883 536

E-mail: [email protected]

(Received 29 June 2006, revised 9 August

2006, accepted 15 August 2006)

doi:10.1111/j.1742-4658.2006.05468.x

FOXM1c transactivates the c-myc promoter via the P1 and P2 TATA boxes

using a new mechanism. Whereas the P1 TATA box TATAATGC requires

its sequence context to be FOXM1c responsive, the P2 TATA box TATA￾AAAG alone is sufficient to confer FOXM1c responsiveness to any minimal

promoter. FOXM1c transactivates by binding to the TATA box as well as

directly to TATA-binding protein, transcription factor IIB and transcrip￾tion factor IIA. This new transactivation mechanism is clearly distinguished

from the function of FOXM1c as a conventional transcription factor. The

central domain of FOXM1c functions as an essential domain for activation

via the TATA box, but as an inhibitory domain (retinoblastoma protein￾independent transrepression domain and retinoblastoma protein-recruiting

negative regulatory domain) for transactivation via conventional FOXM1c￾binding sites. Each promoter with the P2 TATA box TATAAAAG is

postulated to be transactivated by FOXM1c. This was demonstrated for the

promoters of c-fos, hsp70 and histone H2B⁄ a. A database search revealed

almost 300 probable FOXM1c target genes, many of which function in

proliferation and tumorigenesis. Accordingly, dominant-negative FOXM1c

proteins reduced cell growth approximately threefold, demonstrating a pro￾liferation-stimulating function for wild-type FOXM1c.

Abbreviations

BRE, TFIIB recognition element; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DPE, downstream promoter element;

EDA, essential domain for activation; EMSA, electrophoretic mobility shift assay; FKH, forkhead domain; GST, glutathione S-transferase;

GTF, general transcription factor; Inr, initiator; NE, neutrophile elastase; NLS, nuclear localization signal; NRD, negative regulatory domain;

OHT, 4-hydroxy-tamoxifen; PIC, preinitiation complex; RB, retinoblastoma protein; SV40, simian virus 40; TAD, transactivation domain; TAF,

TBP-associated factor; TBP, TATA-binding protein; TFIIB, transcription factor IIB; TK, thymidine kinase; TPA, 12-O-tetradecanoylphorbol-13-

acetate; TRD, transrepression domain.

FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4645

transcription factor the splice variant FOXM1c (MPP2)

binds to FOXM1-specific DNA sequences via its fork￾head domain and transactivates via its strong acidic

transactivation domain (TAD) [29–31]. This strong

TAD can be kept almost inactive by two different

inhibitory domains. The N-terminus functions as a

specific negative regulatory domain (NRD), named

NRD-N, which completely inhibits the TAD by directly

binding to it. The central domain functions as a retino￾blastoma protein (RB)-independent transrepression

domain (TRD) [29–31] and as RB-recruiting NRD-C

[31].

Core promoters and basal transcription complexes

were initially thought to be interchangeable at will, but

are now viewed as active participants in gene regula￾tion. Their diversity makes essential contributions to

the specificity and variability in combinatorial gene

regulation [32–34]. Core promoter elements are the

TATA box, the initiator (Inr), the downstream promo￾ter element (DPE), motif ten element (MTE) and the

transcription factor IIB (TFIIB) recognition element

(BRE). None of these elements is obligatory and sev￾eral different combinations are operational. Enhancers

can target certain core promoter elements so that their

activating effect is limited to genes with these elements

[32–35]. Basal transcription complexes are not uniform

because of TATA-binding protein (TBP)-related fac￾tors and alternative TBP-associated factors (TAFIIs)

[36,37]. It is believed that the basal transcription com￾plex can adopt different conformations on different

core promoters and that different core promoters can

determine different rate-limiting steps in preinitiation

complex (PIC) assembly and transcription initiation, as

well as different reinitiation rates [32–34,38–48].

TBP plays a central role in the recognition of TATA

box promoters. The C-terminal ⁄ core region of TBP

has a saddle-like structure: its concave underside binds

to DNA; the convex upper surface binds to a large

variety of TAFIIs, general trancription factors (GTFs),

transcription factors, coactivators and general cofac￾tors [38,49,50]. TBP binds to the minor groove of the

TATA box, thereby bending the DNA 80 towards the

major groove, unwinding the DNA by 120 and kink￾ing the TATA box at both ends by intercalation of

two phenylalanine residues. TFIIA interacts with the

N-terminal TBP stirrup, which is orientated towards

the 3¢-end of the TATA box, and with TBP helices H1

and H2. TFIIB interacts with the C-terminal TBP stir￾rup, which is orientated towards the 5¢-end of the

TATA box, and with TBP helix H1¢ [38,39,51].

The PIC can be assembled in a stepwise fashion in

reconstituted in vitro systems [38,39]. In vivo, PIC

assembly may vary among core promoters between

two extremes: (a) the stepwise assembly of individual

GTFs, and (b) recruitment of the complete holo￾enzyme in one step [45]. However, PIC assembly will

always require at least two separate steps, namely

TFIID ⁄TFIIA binding and TFIIB⁄ Pol II binding [46].

Here, we describe a new transactivation mechanism

by which FOXM1c transactivates the c-myc promoter

via its P1 and P2 TATA boxes. It does so by binding

to the TATA box and directly to TBP, TFIIB and

TFIIA. The P1 TATA box TATAATGC requires its

sequence context to be FOXM1c responsive. In con￾trast, the P2 TATA box TATAAAAG alone is

sufficient to confer FOXM1c responsiveness on any

minimal promoter so that each promoter with this

TATA box is postulated to be transactivated by

FOXM1c as seen for c-fos, hsp70 and histone H2B⁄ a.

In addition to these new FOXM1c target genes, a

database search revealed nearly 300 genes with such a

TATA box sequence, many of which also play a role

in proliferation and tumorigenesis. Accordingly, dom￾inant-negative FOXM1c proteins reduce cell growth by

approximately threefold demonstrating a proliferation￾stimulating function for wild-type FOXM1c.

Results

FOXM1c transactivates the c-myc promoter,

namely the minimal P1 and P2 promoters

Human c-myc promoter was transactivated by wild￾type FOXM1c and significantly more so by the mutant

FOXM1c(189–762) (Fig. 1A), which lacks the negat￾ive-regulatory N-terminus (see below). Therefore,

FOXM1c(189–762) was used in this study. In contrast

to c-myc, FOXM1c(189–762) did not transactivate the

promoters of human c-jun, waf1(p21), ink4a(p16),

murine neutrophile elastase (NE) or the simian virus

(SV)40 early promoter (Fig. 1B; data not shown).

To map the FOXM1c-responsive element, several

c-myc–promoter constructs were analyzed (Fig. 1D).

FOXM1c(189–762) strongly transactivated the P1

and P2 promoters, but not the P0 promoter. Because

all potential FOXM1c-binding sites (C ⁄T-AAA-C ⁄T)

of the c-myc promoter are positioned in the non￾FOXM1c-responsive segment )2486 ⁄)259 (Fig. 1D;

data not shown), common elements of the P1 and

P2 promoters were analyzed for FOXM1c responsive￾ness. The P1 and P2 promoters both possess a

TATA box and a GC-box-type Sp1-binding site.

The Sp1-binding sites )44 (known; position )44

relative to the P1 transcription start site) and )66

(potential; position )66 relative to the P2 transcription

start site), as well as overlapping binding sites for

FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves

4646 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS